This invention relates to methods of amplifying nucleic acids to minimize contamination by products of earlier amplification reactions. More particularly, it relates to methods of using nucleic acid labels that inhibit further amplification of the amplicon.
Detecting specific gene sequences in clinical samples that are associated with disease states or biological conditions is frequently hindered by the low copy number of these gene sequences in the sample. The ability to replicate these gene sequences to improve sensitivity has revolutionized modern molecular genetics. There are currently many different methods for amplifying nucleic acids in samples to improve assay sensitivity, such as: polymerase chain reaction (PCR) (U.S. Pat. Nos. 4,683,195 and 4,683,202); ligase chain reaction (LCR); nucleic acid sequence-based amplification (NASBA) (U.S. Pat. Nos. 5,409,818 and 5,554,517); strand displacement amplification (SDA); and transcription-medicated amplification (TMA).
Many of these methods are capable of providing more than one billion copies of a single target nucleic acid in a very short time. Accordingly, one of the principle problems of using amplification technologies is that they are susceptible to contamination by exogenous nucleic acids. Although the latter can be controlled using careful laboratory techniques, the former source of contamination is hard to avoid in laboratories that repetitively amplify the same target sequences. In either case, this exogenous nucleic acid may be amplified along with the target nucleic acid in a clinical sample, which may lead to erroneous results.
Many different protocols have been developed in the past several years to prevent carryover contamination. Some of these protocols involve chemical, photochemical and/or enzymatic methods to inactivate the amplicons to prevent them from serving as templates in subsequent amplification reactions. When such methods are combined with appropriate laboratory techniques, the frequency of contamination-associated false-positive results is reduced. Since many of these types of decontamination protocols interfere with the amplification reaction, they must be carried out after amplification has been completed.
One of the more recently described methods for preventing contamination involves the use of UV irradiation to photochemically modify amplicon nucleotide bases. Such irradiation in the presence of certain isopsoralen derivatives forms cyclobutane adducts with pyrimidine bases, and the nucleic acids with these modified bases are no longer capable of serving as templates for subsequent PCR (G. D. Cimino et al., Nucleic Acid Research, 19(1):99-107 (1990)). However, this method has been described as only being useful when carried out after amplification has been completed, since these base reactions are non-specific and the reactants may interfere with the integrity of the target nucleic acid and other reaction components (R. Y. Walder et al., Nucleic Acids Research, 21(18):4339-4343 (1993)). Moreover, most of these currently used methods are adapted for use in PCR. Thus, it is not well established that such methods are equally as effective in other types of amplification reactions. Nor have decontamination protocols been specifically designed to be carried out during any stage of the amplification reaction.
Detection of the amplified nucleic acids involves the use of a labeling compound or compounds that can be measured and quantified. Many such labeling compounds are well known in the art. However, every additional step in the amplification reaction introduces additional reagent costs and assay time. Recently, methods have been develop that allow for simultaneous labeling and decontamination using reagents that are capable of serving both purposes.
Accordingly, there is a need to provide for improved decontamination reagents and protocols that are adapted for use before the amplification reaction has been completed, and that are suitable for simultaneous decontamination and labeling.
The present invention provides compositions that are useful for labeling and decontaminating a nucleic acid amplification reaction product, also referred to herein as an xe2x80x9campliconxe2x80x9d. Such compositions comprise xe2x80x9cLACsxe2x80x9d, which are covalent or noncovalent complexes of a binding ligand, a binding enhancer and a label. As described herein, the binding ligand is a chemical moiety that binds to the amplicon and that, when activated by light, forms at least one covalent bond therewith. Also as described herein, the binding enhancer is a chemical moiety that has a specific affinity for nucleic acids when compared to its affinity for the non-nucleic acid components of amplification reactions. As provided herein, the label is a detectable chemical moiety, such as a fluorophore, a chemiluminescent label or other chromophore.
In one aspect of the present invention, the binding ligand is either an intercalator compound, such as a furocoumarin or a phenanthridine, or a nonintercalator compound, such as a benzimide, a netropsin or a distamycin. When the binding ligand is an intercalator compound, in a preferred embodiment, it is an angelicin derivative.
In another aspect of the present invention, the binding enhancer is also either an intercalator compound or a nonintercalator compound, such as an oligo pyrrole, a phenyl indole, a nucleic acid or a protein.
In one embodiment, the LAC of the present invention is a complex of at least two intercalator moieties and a label.
Another aspect of the present invention is a method for labeling or decontaminating a nucleic acid amplification reaction product comprising the steps of preparing a nucleic acid amplification reaction mixture, contacting the mixture with the compositions just described, and exposing the mixture to light of an appropriate length of time and wavelength to cause the binding ligand to become covalently attached to the nucleic acid amplification reaction product.
Other aspects of the invention are described throughout the specification.
The present invention relates to a method of amplifying a target analyte nucleic acid to produce multiple copies of the target, i.e. the nucleic acid reaction products of the amplification reaction which are also referred to as xe2x80x9campliconsxe2x80x9d, and contacting the amplicons with a photoreactive compound, or xe2x80x9clight-activated compoundxe2x80x9d (xe2x80x9cLACxe2x80x9d) that serves the dual purpose of labeling and xe2x80x9cdeactivatingxe2x80x9d the amplicons. The present invention also relates to compositions comprising such LACs. By xe2x80x9cdeactivatingxe2x80x9d, it is meant that the photo-activated amplicons can no longer be amplified. In particular, the LAC is added to the amplification reaction before, during or after the nucleic acid amplification reaction. After the amplification reaction is completed, the reaction mixture is exposed to light of an appropriate wavelength to cause the labeling compound to become covalently linked to the amplicon. Thereafter, the amplicon is incapable of serving as a template for polymerization and thus prevented from contaminating subsequent amplification reactions.
Definitions
The following definitions are provided to further describe various aspects of the preferred embodiments of the present invention.
The term xe2x80x9camplificationxe2x80x9d is used to refer to a method for exponentially duplicating a target analyte nucleic acid in a sample to improve assay sensitivity. As described herein, many different methods for amplifying nucleic acids are known in the art. It should be understood that the particular amplification method employed in the practice of the present invention can vary depending on the type of target analyte, the type of sample, the desired sensitivity, and the like. The selection and performance of such amplification methods are not within the scope of the present invention.
The term xe2x80x9cbinding ligandxe2x80x9d is used to refer to a compound that has an affinity for nucleic acids, such that it forms a reversible complex with nucleic acids, and is capable of being activated upon the application of an appropriate wavelength of light to form a covalent bond with the nucleic acids.
The term xe2x80x9cbinding enhancerxe2x80x9d is used to refer to a chemical moiety that has a specific affinity for nucleic acids.
The term xe2x80x9clabelxe2x80x9d is used to refer to any chemical group or moiety having a detectable physical property or any compound capable of causing a chemical group or moiety to exhibit a detectable physical property, such as an enzyme that catalyzes conversion of a substrate into a detectable product. The term xe2x80x9clabelxe2x80x9d also encompasses compounds that inhibit the expression of a particular physical property. The label may also be a compound that is a member of a binding pair, the other member of which bears a detectable physical property.
The term xe2x80x9cnucleic acid(s)xe2x80x9d is used to refer to deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) in any form, including inter alia, single-stranded, duplex, triplex, linear and circular forms. It also includes polynucleotides, oligonucleotides, chimeras of nucleic acids and analogues thereof. The nucleic acids described herein can be composed of the well-known deoxyribonucleotides and ribonucleotides composed of the bases adenosine, cytosine, guanine, thymidine, and uridine, or may be composed of analogues or derivatives of these bases. Additionally, various other oligonucleotide derivatives with non-conventional phosphodiester backbones are also included herein, such as phosphotriester, polynucleopeptides, methylphosphonate, phosphorothioate, polynucleotides primiss and the like.
The term xe2x80x9ctarget analytexe2x80x9d is used to refer to the particular nucleic acid that a sample is suspected of containing. Such analytes include nucleic acids in biological samples, research materials, environmental samples, bodily fluids, and may be unpurified or purified using known methods. For an example of types of target analytes, see U.S. Pat. No. 5,792,614.
LAC Structure and Function
The LACs that are useful in the practice of the present invention are designed to be compatible with any target amplification protocol, and can be added to the amplification reaction before, during or after the initiation of the amplification reaction. In particular, the LACs of the present invention are designed to have an enhanced affinity for nucleic acids so that they will efficiently bind to nucleic acid in the presence of other sample and amplification reaction constituents, such as proteins, lipids, enzymes, multivalent cations, etc. Such enhanced affinity permits a lower concentration of LAC to be necessary for efficient decontamination and labeling. Thus, a noninhibitory amount of LAC can be added to the amplification reaction mixture before amplification has taken place. Thereafter, the LAC can be exposed to light to effect simultaneous decontamination and labeling.
In a preferred embodiment, the LACs of the present invention are complexes comprising a binding ligand (xe2x80x9cbinding ligandxe2x80x9d), a nucleic acid binding enhancer (xe2x80x9cbinding enhancerxe2x80x9d), and a label. Such complexes can be either linear or branched. An example of a linear complex is given below:
BINDING LIGANDxe2x80x94BINDING ENHANCERxe2x80x94LABEL
In this example, the binding enhancer serves as the linker between the binding ligand and the label. Alternatively, an example of a branched complex is given below: 
In this example, the binding ligand, binding enhancer and label are all interconnected to one another.
Binding Ligands
The binding ligand of the present invention is preferably any photoreactive chemical moiety that reversibly binds to nucleic acids and forms at least one covalent bond with the nucleic acid when exposed to light of an appropriate wavelength. In a preferred embodiment, the photoreactive binding ligand is an intercalator compound (i.e. a compound that interposes itself between the nucleotide bases of a nucleic acid helix). Suitable intercalator binding ligands include, inter alia, furocoumarins and phenanthridines. For binding to DNA, aminomethyl psoralen, aminomethyl angelicin and aminoalkyl ethidium or methidium azides are somewhat useful. Although these compounds preferentially bind to double-stranded DNA, conditions can be employed to denature the DNA to avoid simultaneous interaction of these compounds with two strands.
In order to preserve the ability of the labeled amplicon to participate in hybridization reactions, it is desirable to use binding ligands that react with a single nucleic acid strand. Accordingly, preferred binding ligands are xe2x80x9cmonoadductxe2x80x9d forming compounds such as isopsoralen or other angelicin derivatives, such as 4xe2x80x2-aminomethyl, 4,5xe2x80x2-dimethyl angelicin, 4xe2x80x2-aminomethyl 4,5xe2x80x2,8-trimethyl psoralen, 3-carboxy-5- or 8-amino- or hydroxy-psoralen, as well as mono- or bis-azido aminoalkyl methidium or ethidium compounds. For examples of other photoreactive intercalators, see U.S. Pat. No. 4,734,454. Nonintercalating compounds, such as diamidinoindophenol-bis-benzimidazoles, which are commonly known as Hoechst 33258 and 33342, and other benzimides, netropsins and distamycins can also be used in the present invention. Preferred photoreactive binding ligands are the monoadduct forming psoralens and isopsoralens.
Binding Enhancers
The LACs of the present invention also comprise a nucleic acid binding enhancer (xe2x80x9cbinding enhancerxe2x80x9d), which serves to enhance the affinity of the LAC for nucleic acids above that exhibited with the binding ligand alone. Accordingly, binding enhancers tend to have a specific affinity for nucleic acids when compared to non-nucleic acid sample/reaction constituents. The binding enhancer may be the same as or different from the binding ligand. In other words, the binding ligand and the binding enhancer may each be an intercalator, wherein one of the two is a monoadduct-forming species, and the other is present to enhance binding by this monoadduct-forming species. Examples of such xe2x80x9cdual rolexe2x80x9d binding ligands are described in J. B. Chaires, et al., J. Med. Chem, 40:261-266 (1977). Therein, it has been described that binding of a bis-intercalating anthracycline antibiotic reached as high as 1011 at 20xc2x0 C. It was also shown that the affinity of a similar monointercalator is not above 107 (J. B. Chaire, et al., Biochemistry, 35:2047-2053 (1996).
The binding enhancer can also be a non-intercalating compound. There are many nonintercalating nucleic acid binding molecules known in the art. A bis-benzimidazole derivative commonly known as Hoechst 33258 has shown affinity as high as 3.2xc3x97108 molexe2x88x921. (Haq et al., J. Mol. Biol., 271:244-257(1997).) Other non-intercalating binding enhancers are oligo pyrroles, phenyl indole derivatives and such molecules. These molecules do not bind nucleic acids only on the basis of positive charge. Other suitable binding enhancers bind nucleic acids on the basis of hydrogen bond formation, hydrophobic interaction in the groove and other nonionic interactions that give rise to high affinity reactions with nucleic acids. In general, preferred binding enhancers will exhibit an affinity for nucleic acids in an amount equal to or greater than 1xc3x97104 molexe2x88x921. Other suitable binding enhancers include nucleic acids having a specific affinity for other nucleic acids, such as would be expected if the binding enhancer had a nucleic acid sequence complementary to that of the amplicon target nucleic acid. Yet other suitable binding enhancers include proteins that have a specific binding affinity for nucleic acids.
Not every compound capable of forming an electrostatic bond with a negatively charged nucleic acid can serve as a binding enhancer. For example, polycations such as polyaminies are generally not suitable for use in the present invention because of their inability to specifically bind to nucleic acids in crude samples and in the presence of amplification reaction components. For example, such positively charged compounds will nonspecifically bind to all anionic macromolecules present in the sample, and not just to nucleic acids. In addition, the binding enhancer should be capable of specifically binding to nucleic acids in the presence of 10 to 20 mM magnesium, which is typically required for most amplification reactions. At this concentration, compounds that bind to nucleic acids solely on the basis of electrostatic interactions would not form stable complexes with nucleic acids and thus would require addition of a greater concentration of LAC for efficient labeling.
Labels
In the practice of the present invention, the binding ligand is either directly or indirectly linked to a label. Such attachment can be either covalent or ionic, so long as it is stable under the conditions in which the LAC is employed. Chemical attachments can be accomplished by any of a variety of well known methods. For example, if the binding ligand contains or is derivatised to contain an available carboxyl group and the label contains or is derivalized to contain an available amino group, the two can be reacted together to form an ester linkage. By xe2x80x9cavailablexe2x80x9d, it is meant that the formation of a linkage will not interfere with the functioning of the label (i.e. its ability to be detected or to catalyze a detectable reaction) or the ligand (i.e. it""s ability to bind nucleic acids.)
Particularly useful labels are enzymes, enzyme substrates, fluorophore, radiocsotopic compounds, chromophores, magnetically responsive compounds, antibody epitope-containing compounds, haptens, and the like.
Linkers
The binding ligand, binding enhancer, and label can also be indirectly attached via a linker. The linkers that are useful in the practice of the present invention are specifically designed to promote efficient binding of the binding ligand to the nucleic acids and functioning of the label attached thereto. They accomplish this by providing adequate physical separation between the two components of the LAC to prevent interference of one by the other. The use of linkers is described generally in U.S. Pat. Nos. 4,582,789 and 5,026,840. Certain compounds can serve the dual role of a binding enhancer and a linker. For example, linkers can be constructed from positively charged compounds, such that they enhance binding with negatively charged nucleic acids. However, in order for the linker to also serve as a binding enhancer, it is necessary for it to have a specific affinity for nucleic acids, and not just a non-structure specific electrostatic affinity for negatively charged compounds. Accordingly, the polyalkylamine linkers described in U.S. Pat. No. 5,026,840 are specifically excluded by the present invention as binding enhancers, although they may still be suitable for use as linkers.
In a preferred embodiment, a bifunctional linker is used that is capable of reacting with both the nucleic acid binding moiety and the label to form a chemical bridge therebetween. However, in an alternate embodiment, a multifunctional linker may be employed, to which the binding ligand, the binding enhancer and the label can all be attached to form a xe2x80x9cbranchedxe2x80x9d complex. Such complex formats and chemical reactions for forming these types of complexes are well known in the art.
Formulation and Use of LACs
The LACs of the present invention are useful for labeling and deactivating the amplicon products of nucleic acid amplification protocols. As such, they are generally prepared as an aqueous solution in an appropriate liquid medium at a concentration of around 10 micromolar to 10 millimolar. An aliquot of such solution is added to the amplification reaction mixture. The final preferable concentration of LAC in such a mixture is between about 1 micromolar and 1 millimolar, and more preferably the range is between about 0.01 micromolar and 0.1 millimolar. Depending on the LAC""s affinity, concentrations lower than 0.01 micromolar would also function in instances where the affinity of the LAC for the nucleic acid was high. For deactivation and labeling, less than one LAC per twenty nucleotides is sufficient. Accordingly, efficiency of labeling can easily be achieved by using less than a ten to one ratio of LAC to nucleic acid. Determining the appropriate ratio would be a matter of routine optimization.
The aqueous medium for the LAC solution can be water or a buffer solution, the pH of which should preferably be such that the compound is stable. Such stability can be determined or easily assessed. For example, if a LAC with acridinium ester is used, the pH should not be alkaline. Otherwise, for most compounds, the pH can be between 3 and 12. Preferably, the pH is between 5 and 11 for compounds that have no acridinium ester or other alkali-hydrolysable moiety. Appropriate concentrations can also be determined by measuring the affinity of a specific LAC for nucleic acids and optimizing the binding-conditions by methods known in the art. Such affinity values can be used to determine what concentration would produce a labeling efficiency of choice.
Another feature of the present invention is that the LAC, once light-activated, prevents the amplicon from being amplified in a subsequent amplification reaction. Accordingly, it is necessary to add sufficient LAC to the amplification reaction to essentially completely xe2x80x9cdeactivatexe2x80x9d the labeled amplicons, without inhibiting subsequent detection reactions which may or may not depend on the amplicon""s ability to hybridize. One of skill in the art could easily determine the least amount and greatest amount of LAC to be added to any given amplification reaction by carrying out simple optimization studies on a research scale.
The LACs of the present invention can be added to the amplification reaction before or after amplification has been carried out. It is generally not suitable for the LAC formulation to be added during the amplification reaction, since it is important to keep the reaction mixture closed to the environment to avoid contamination. However, if an appropriate reaction vessel is designed wherein the LAC can be added at some stage after amplification has begun but before it has been stopped without exposing the reaction mixture to the environment, one could easily envision that the LAC could be added during the amplification reaction. Since many amplification reactions are cyclic, by using the term xe2x80x9cduring the amplification reactionxe2x80x9d, it is meant that the LAC is added after the first amplification cycle, but before the last cycle.
After addition, the LAC is mixed into amplification reaction mixture. Such mixing can be done under a wide variety of conditions of time, temperature and types of mixing devices. Since these compounds are in aqueous solution, mixing by shaking after addition for more than 15 seconds should be adequate. If the sample solution is viscous, a longer period may be needed. Mixing time can easily be determined by one skilled in the art. Usually, such mixing can be carried out at ambient temperature, but an elevated temperature may also be used, so long as the integrety of the LAC is maintainable at this temperature.
To activate the LAC for covalent attachment with the target nucleic acids, the wavelength of choice is dependent on the photoactivatable moeties in LAC. For example, with furocoumarines like angelicin, wavelengths between 300 and 370 nm are preferred, and wavelengths between 320 and 350 nm are more preferred. For compounds like azideothidium, a longer wavelength light source may be desirable. Appropriate activation wavelengths can be found in the scientific literature for most photoactivatable intercalating compounds, or such wavelengths can be easily determined by one skilled in the art.